Actinobacillus succinogenes shuttle vector and methods of use

ABSTRACT

An  Actinobacillus succinogenes  plasmid vector which provides a means to overexpress proteins in  A. succinogenes.  The plasmid can be transformed efficiently by electroporation, and replicates in a stable manner in  A. succinogenes.  The plasmid comprises at least one marker gene, operably linked to a first promoter functional in  Actinobacillus succinogenes,  an origin of replication functional in  Actinobacillus succinogenes,  a second promoter isolated from  Actinobacillus succinogenes,  and a cloning site downstream from the second promoter. Plasmids pLGZ901, pLGZ920, pLGZ921, and pLGZ922 are disclosed. The pckA gene polypeptide sequence and nucleic acid sequence of  Actinobacillus succinogenes,  including the promoter and ribosome binding site, is disclosed. Furthermore, a method for producing a recombinant  Actinobacillus succinogenes  is described, including a method of transformation. Additionally, a recombinant  Actinobacillus succinogenes  is disclosed and a method for producing succinate utilizing this recombinant  Actinobacillus succinogenes  is described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Provisional application Ser. No.60/492,804, filed Aug. 6, 2003.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This research was supported by grants from the USDA (#00-34189-9045) andfrom the National Science Foundation NSF (#BES-0224596). The U.S.government has certain rights to this invention.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates generally to plasmid vectors, and moreparticularly to Actinobacillus succinogenes plasmid vectors. The presentinvention further relates to overexpression of proteins in A.succinogenes and to engineered A. succinogenes strains which have theplasmids introduced into them.

(2) Description of the Related Art

Succinate has many industrial fine chemical uses. Succinate can also beused as an intermediary commodity chemical feedstock for producing bulkchemicals. Its greatest market potential might lie in its use asfeedstock to produce stronger-than-steel plastics, biodegradablechelators, and green solvents. Most of the 17,000 tons (15,400 metrictons) of succinate sold per year are produced petrochemically frommaleic acid. Succinate is also produced as a fine chemical byfermentation from glucose. For fermentation to be competitive inproducing succinate as a commercial chemical, the overall productioncost should be lowered from one (1) dollar per pound ($2.20 per kg) toapproximately twenty (20) cents per pound (44 cents per kg).

U.S. Pat. No. 5,143,833 to Datta et al. teaches a method for producingsuccinic acid by growing a succinate producing Anaerobiospirillumsucciniciproducens microorganism under specific conditions.

U.S. Reissued Patent No. RE37,393 to Donnelly et al. teaches a methodfor isolating succinic acid producing bacteria by increasing the biomassof an organism which lacks the ability to catabolize pyruvate and thengrowing the biomass in glucose-rich medium under an anaerobicenvironment to enable pyruvate-catabolizing mutants to grow. By usingthis method, Donnelly provides a mutant E. coli that produces highamounts of succinic acid. The mutant E. coli was derived from a parentwhich lacked the genes for pyruvate formate lyase and lactatedehydrogenase.

U.S. Pat. No. 6,455,284 and U.S. Pat. Application Publication No.2003/0087381, both to Gokarn et al. teach metabolic engineering toincrease the carbon flow toward oxaloacetate to enhance production ofbulk biochemicals, such as lysine and succinate, in bacterial andindustrial fermentations. The carbon flow is redirected by geneticallyengineering bacteria to overexpress the enzyme pyruvate carboxylase.

U.S. Pat. Application Publication No. 2003/0113885 to Lee et al. teachesa novel microorganism, Mannheimia sp. 55E, capable of producing organicacids and a process using the microorganism for producing organic acidsthrough anaerobic and aerobic incubations.

U.S. Pat. No. 6,420,151 to Eikmanns, et al. teaches an isolated nucleicacid from coryneform bacteria which encodes a phosphoenolpyruvatecarboxykinase which is involved in production of succinate.

While the above methods can be used to produce succinate, Actinobacillussuccinogenes is still the best succinate producer known. Actinobacillussuccinogenes is a gram-negative capnophilic, anaerobic bacillus thatbelongs to Pasteurellaceae. A. succinogenes produces up to one hundred(100) grams per liter of succinate in optimized conditions. Much efforthas been spent on engineering Escherichia coli strains to produce highsuccinate amounts, however none of the engineered E. coli strainssurpassed A. succinogenes for succinate production. Carbon flow isstringently regulated in microorganism metabolism, including carbon fluxtowards oxaloacetate. Overcoming this control of carbon flux willpossibly improve the yields of desirable products, such as succinate.

Previously, no genetic tools had been tested or developed that could beused for engineering A. succinogenes into an industrialsuccinate-producing strain. Therefore, it would be desirable to have ameans to genetically engineer A. succinogenes to overproduce succinate.However, there is not yet a means for constructing recombinant A.succinogenes. The present invention provides a means for constructingrecombinant A. succinogenes using a plasmid for the expression ofproteins in A. succinogenes.

SUMMARY OF THE INVENTION

The present invention provides a plasmid comprising at least one markergene operably linked to a first promoter functional in Actinobacillussuccinogenes, an origin of replication functional in Actinobacillussuccinogenes, a second promoter isolated from Actinobacillussuccinogenes, and a cloning site downstream from the second promoter.

In further embodiments, the marker gene confers antibiotic resistance toActinobacillus succinogenes. In further embodiments, the antibiotic isselected from the group consisting of ampicillin, chloramphenicol,tetracycline, and erythromycin.

In further embodiments, the second promoter is from the pckA gene ofActinobacillus succinogenes. In some embodiments, the second promotercomprises substantially the nucleic acid sequence set forth in SEQ IDNO: 21 from between about nucleotide 25 and nucleotide 255. In furtherstill embodiments, the second promoter provides a ribosome binding sitecomprising the nucleotide sequence AGGTG. In further still embodiments,the plasmid further comprises a ColE1 origin of replication. In furtherstill embodiments, the cloning site comprises one or more restrictionendonuclease cleavage sites.

The present invention also provides a plasmid which is pLGZ901 and aplasmid which is pLGZ920 (ATCC deposited on Aug. 3, 2004).

The present invention further provides a polypeptide which comprises anamino acid sequence substantially similar to the amino acid sequence setforth in SEQ ID NO. 22. The present invention still further provides anucleic acid which comprises a nucleotide sequence substantially similarto the nucleotide sequence set forth in SEQ ID NO: 21.

The present invention further provides a method for producing arecombinant Actinobacillus succinogenes comprising providing a plasmidcomprised of at least one marker gene operably linked to a firstpromoter functional in Actinobacillus succinogenes, an origin ofreplication for the plasmid functional in Actinobacillus succinogenes, asecond promoter isolated from Actinobacillus succinogenes, and a cloningsite for a nucleic acid downstream of the second promoter, transformingan Actinobacillus succinogenes with the plasmid, and selecting therecombinant Actinobacillus succinogenes from non-transformedActinobacillus succinogenes.

In some embodiments of the method, the transformation iselectroporation. In some embodiments of the method, the marker geneconfers antibiotic resistance to the recombinant Actinobacillussuccinogenes. In further embodiments of the method, the antibiotic isampicillin, tetracycline, or chloramphenicol. In some embodiments of themethod, the recombinant Actinobacillus succinogenes is selected fromnon-transformed Actinobacillus succinogenes by culturing in the presenceof the antibiotic. In some embodiments of the method, the secondpromoter is from the pckA gene of Actinobacillus succinogenes. In someembodiments of the method, the second promoter comprises substantiallythe nucleic acid sequence set forth in SEQ ID NO: 21 from between aboutnucleotide 25 and nucleotide 255. In some embodiments of the method, thesecond promoter provides a ribosome binding site comprising thenucleotide sequence AGGTG. In some embodiments of the method, theplasmid further includes a ColE1 origin of replication. In someembodiments of the method, the plasmid is pLGZ901 or pLGZ920.

The present invention further provides a recombinant Actinobacillussuccinogenes comprising a plasmid capable of autonomous replication inActinobacillus succinogenes which comprises at least one selectablemarker gene and the Actinobacillus succinogenes pckA gene. In someembodiments of the recombinant Actinobacillus succinogenes, the pckAgene comprises the nucleic acid sequence set forth in SEQ ID NO: 21. Insome embodiments of the recombinant Actinobacillus succinogenes, theplasmid is pLGZ902. In some embodiments of the recombinantActinobacillus succinogenes, the marker gene confers ampicillin,tetracycline, or chloramphenicol resistance to the recombinantActinobacillus succinogenes.

The present invention further provides a method for producing succinatecomprising providing a recombinant Actinobacillus succinogenescomprising a plasmid capable of autonomous replication in Actinobacillussuccinogenes which comprises at least one selectable marker gene and arecombinant gene expressed under the control of the Actinobacillussuccinogenes pckA promoter, providing a growth medium for culturing therecombinant Actinobacillus succinogenes, and culturing the recombinantActinobacillus succinogenes in the growth medium to produce thesuccinate.

In some embodiments of the method the promoter comprises substantiallythe nucleic acid sequence set forth in SEQ ID NO: 21 from between aboutnucleotide 25 and nucleotide 255. In some embodiments of the method theplasmid is pLGZ901, and in further embodiments the plasmid is pLGZ920.In some embodiments of the method the marker gene confers ampicillin,tetracycline, or chloramphenicol resistance to the recombinantActinobacillus succinogenes. In some embodiments of the method thegrowth medium comprises ampicillin, tetracycline, or chloramphenicol.

In further embodiments of the method the recombinant Actinobacillussuccinogenes is a mutant strain in which an Actinobacillus succinogenesgene which inhibits succinate production comprises a deletion. Infurther embodiments of the method the Actinobacillus succinogenes genefurther comprises a selectable marker under the control of theActinobacillus succinogenes pckA promoter. In further embodiments of themethod the selectable marker gene confers resistance to an antibioticselected from the group consisting of chloramphenicol and tetracyclineto the mutant Actinobacillus succinogenes strain.

The present invention further provides a recombinant microorganismcomprising the nucleic acid which comprises a nucleotide sequencesubstantially similar to the nucleotide sequence set forth in SEQ ID NO:21.

The present invention further provides a plasmid which is pLGZ921, and aplasmid which is pLGZ922.

OBJECTS

Therefore, it is an object of the present invention to provide a methodfor producing recombinant Actinobacillus succinogenes.

It is a further object of the present invention to provide a plasmidwhich replicates in A. succinogenes.

It is further still an object of the present invention to provide arecombinant A. succinogenes which overexpresses proteins.

These and other objects will become increasingly apparent by referenceto the following description and the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the nucleotide sequence SEQ ID NO: 21 of A.succinogenes pckA and deduced amino acid sequence SEQ ID NO: 22 ofPEPCK. Two pairs of putative −35 and −10 promoter regions areunderlined. The putative ribosome binding site (RBS) is in bold. Thestop codon is indicated by three asterisks. The GenBank accession numberfor the nucleotide sequence is AY308832.

FIG. 2 illustrates β-Galactosidase activity in A. succinogenes 130Zgrown in medium A.

FIG. 3 illustrates β-Galactosidase activity in A. succinogenes 130Zgrown in TS broth.

FIG. 4 illustrates the stability of plasmids pLGZ901 and pLGZ920 in A.succinogenes and E. coli.

FIG. 5 illustrates the physical map of the pGZRS-1 plasmid. The dashedline represents the minimum region required for replication in A.pleuropneumoniae.

FIG. 6 illustrates the physical map of the pLGZ901 plasmid. The originof replication functional in A. succinogenes, the cloning site (MCS),the A. succinogenes promoter, and the bla-Tn3 marker gene are shown.

FIG. 7 illustrates the physical map of the E. coli-A. succinogenesshuttle vector pLGZ920. The ColE1 origin of replication, the origin ofreplication functional in A. succinogenes, the cloning site (MCS), theA. succinogenes promoter, and the bla-Tn3 marker gene are shown.

FIG. 8 illustrates the physical map of the pLGZ920 plasmid with aninserted pckA gene. The ColE1 origin of replication, the origin ofreplication functional in A. succinogenes, the cloning site (MCS), theA. succinogenes promoter, the pckA gene, and the bla-Tn3 marker gene areshown.

FIG. 9 illustrates the time for plasmid-harboring A. succinogenes toform colonies on TS agar at 37° Celsius in the presence of tetracyclineor chloramphenicol.

DETAILED DESCRIPTION OF THE INVENTION

All patents, patent applications, government publications, governmentregulations, and literature references cited in this specification arehereby incorporated herein by reference in their entirety. In case ofconflict, the present description, including definitions, will control.The plasmids pLGZ901, pLGZ920, pLGZ921, and pLGZ922 are available uponrequest from Michigan State University.

Definitions for the following terms are provided to promote a furtherunderstanding of the present invention.

The term “marker” refers to sequences which encode a gene product,usually an enzyme, that inactivates or otherwise detects or is detectedby a compound in the growth medium. For example, the inclusion of amarker sequence can render the transformed cell resistant to anantibiotic, or it can confer compound-specific metabolism on thetransformed cell. Marker genes can confer resistance to antibioticsincluding, but not limited to, ampicillin, chloramphenicol,tetracycline, and erythromycin.

The term “pLGZ901” refers to the plasmid construct as represented byFIG. 6. The plasmid is available upon request from Michigan StateUniversity.

The term “pLGZ920” refers to the plasmid construct as represented byFIG. 7. The plasmid is available upon request from Michigan StateUniversity, and has been deposited under the terms of the BudapestTreaty at the ATCC, Manassas, Va. on Aug. 3, 2004, with accession number______.

The term “promoter” refers to a DNA fragment to which ribonucleic acidpolymerase binds to initiate the transcription of nucleic acid sequenceslinked to the promoter.

A promoter is “operably linked” to a nucleic acid sequence if it can beused to control or regulate transcription of the nucleic acid sequence.

The term “origin of replication” refers to a nucleic acid sequence whichis necessary to allow replication of a plasmid within an organism.

The term “cloning site” refers to a region which allows for theinsertion of desired nucleic acid sequences. Typically, the cloning sitecomprises one or more restriction endonuclease recognition sites.Cloning sites include, but are not limited to, multiple cloning sites orpolylinkers.

The term “ribosome binding site” refers to a short nucleotide sequenceto which the ribosome binds upon the transcribed ribonucleic acid.

The term “transformation” means the process of introducing DNA into anorganism which changes the genotype of the recipient organism.

The term “PEPCK” refers to phosphoenolpyruvate carboxykinase.

The term “vector” refers to a deoxyribonucleic acid which is capable ofreplication and also capable of incorporating desired deoxyribonucleicacid fragments for cloning. Vectors include plasmids, cosmids, phages,and yeast artificial chromosomes.

The term “shuttle vector” refers to a vector able to replicate in twodifferent organisms.

A required step toward A. succinogenes metabolic engineering isdeveloping genetic tools. It is necessary to be able to increase theexpression level of certain enzymes, probably by introducing them onstable, multicopy plasmids, and to shut down some pathways bygeneralized or targeted mutagenesis. One method for producing succinatecan comprise a mutant strain of A. succinogenes in which a selected genewhich minimizes succinate production contains a deletion. Another methodfor producing succinate can comprise both increasing expression levelsof some enzymes by introducing them on plasmids, and shutting down apathway which minimizes succinate production in which a selected genecontains a deletion. The tools needed to achieve this include (i)selection markers, (ii) a transformation and/or conjugation system(s),(iii) a complementation system, and (iv) targeted and generalizedmutagenesis systems.

A plasmid was developed suitable for use in A. succinogenes to expressproteins. First, a replicon that stably replicates in A. succinogeneswas found. A. succinogenes can be transformed by electroporation atreasonably high efficiency by pGZRS-19, pGZRS-19 replicates in a stablemanner in A. succinogenes, and the ampicillin resistance gene carried bypGZRS-19 is expressed in A. succinogenes. These properties made pGZRS-19an excellent starting point to develop an E. coli-A. succinogenesshuttle vector to be used for expressing foreign proteins in A.succinogenes. The plasmid pGZRS-19 was constructed from pGZRS-1,illustrated in FIG. 5, which is a member of the H2 class of Aplplasmids. The pGZRS-1 plasmid is an endogenous Actinobacilluspleuropneumoniae (Apl) 4.3 kilobase plasmid found in the serotype 7strain EL312. (West, S. E., et al., Gene 160:81-86(1995)). The plasmidpGZRS-19 carries the multiple cloning site from pUC19, and the bla genefrom Tn3 under the control of a putative Apl promoter. The plasmidpGZRS-19 replicates in Apl, Escherichia coli, Pasteurella haemolytica,and Haemophilus (Actinobacillus) actinomycetemcomitans. West et al.demonstrated that there is a minimal region required for replication inboth Apl and E. coli, which is within the 1.5 kB PstI- HaeII fragment ofpGZRS-1. We demonstrated that A. succinogenes is transformed byelectroporation at reasonably high efficiency, that pGZRS-19, with thisfragment, replicates in a stable manner in A. succinogenes, and that theampicillin resistance gene carried by pGZRS-19 is expressed in A.succinogenes.

Three steps were required to develop this new vector. (i) A gene that weknew was constitutively expressed at high levels in A. succinogenes(i.e., the pckA gene) was cloned and sequenced. (ii) Its promoter regionand ribosome binding site were subcloned into pGZRS-19. A unique XbaIsite was included immediately downstream of the pckA ribosome bindingsite to facilitate the cloning of foreign genes under control of thepckA promoter. (iii) Finally, the ColE1 origin of replication was addedto the vector to increase its stability in E. coli. High levels ofβ-galactosidase and PEPCK activities detected in cultures of recombinantA. succinogenes strains confirmed that both A. succinogenes and foreignproteins could be expressed in A. succinogenes under control of the A.succinogenes pckA promoter carried by our pGZRS-19-derived vector,pLGZ920.

EXAMPLE 1

Bacterial strains and plasmids used in this study are listed in Table 1.TABLE 1 Plasmid Relevant characteristics References pCR ™ II TOPO TAcloning vector Invitrogen, Carlsbad, CA pCR2.1 TOPO TA cloning vectorInvitrogen pUC19 Amp^(R), lacZα, ColE1 Invitrogen multicopy cloningvector pProEx-1 Amp^(R), ColE1 multicopy cloning [Bolivar, 1978 vector#3323] pBR325 Amp^(R), Cm^(R), Tet^(R), ColE1 cloning Laboratory vectorcollection pGZRS-19 pGZRS-1 replicon, Amp^(R) (Tn3), (30) replicates inAple, Phae, Aact, Replicates in E. coli, pUC18/19 multiple cloning site,lacZα-complementation pGZRS-30 pGZRS-1 replicon, Km^(R) (Tn903), (30)Cm^(R) (Tn9) UB214 Mannheimia haemolytica pMHT1 (17) replicon, Tet^(R)pLGZ901 pGZRS-19 derivative, A. succinogenes This study pckA promoterpLGZ902 pGZRS-19 derivative, A. succinogenes This study pckA genepLGZ903 pGZRS-19 derivative, E. coli lacZα This study under control ofthe A. succinogenes pckA promoter pLGZ920 pGZRS-19 derivative, A.succinogenes This study pckA promoter, pGZRS-1 and ColE1 repliconspLGZ921 pLGZ920 derivative, pBR325 Cm^(R) This study under control oftheA. succinogenes pckA promoter pLGZ922 pLGZ920 derivative, pBR325 Tet^(R)This study under control of the A. succinogenes pckA promoter pGZRS-19/Fusion of pLGZ901 and UB214 This study UB214 plasmids UB214-Amp^(R)UB214 derivative containing Amp^(R) This study from pGZRS-19

E. coli DH5α and JM110 were used for plasmid construction. E. colistrains were grown in LB medium (Sambrook, J., et al., MolecularCloning: a Laboratory Manual, 2^(nd) ed. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989)) supplemented with antibiotics(at 50 μg/ml) when necessary. A. succinogenes 130Z (ATCC 55618) and itsderivative FZ6 were obtained as gifts from MB1 International (Lansing,Mich.). Strain FZ6 is a succinate-overproducing mutant that was selectedby resistance to fluoroacetate (Guettler, M. V., et al., U.S. Pat. No.5,573,931 (1996)). FZ6 produces only pyruvate and succinate asfermentation products from glucose. FZ6 lacks pyruvate-formate lyaseactivity (Park, D. H., et al., J. Bacteriol. 181:2403-2410 (1999)). A.succinogenes strains were grown in Trypticase-Soy (TS) broth(Becton-Dickinson, Cockeyville, Md.) or medium A (per liter: 9 gglucose, 10 g sodium bicarbonate, 5 g yeast extract, 8.5 g NaH₂PO₄.H₂O,and 15.5 g K₂HPO₄). The pH was adjusted to 7.5 with NaOH beforeautoclaving, leading to a pH of 7.0 after autoclaving. The glucose wasadded aseptically after sterilization. A. succinogenes cultures weregrown in butyl-rubber-stopper 18-ml anaerobic tubes and 158-ml serumvials containing 10 ml and 100 ml medium, respectively. When grown onplates, A. succinogenes strains were grown in an anaerobic jar under CO₂atmosphere.

EXAMPLE 2

Antibiotic minimum inhibitory concentrations. To determine thesensitivity of A. succinogenes 130Z to antibiotics, the growth of A.succinogenes 130Z was tested in medium A (liquid medium) containing aseries of antibiotics commonly used in bacteriology. Antibiotics andantibiotic concentrations used are listed in Table 2. One ml of 130Zpreculture was inoculated into 9 ml of medium A containing eachantibiotic and incubated at 37° C. for 7 days. Cell growth was followedby measuring OD₆₆₀. TABLE 2 Antibiotics and antibiotic concentrationsused in this study. Concentration used Concentrations in plates aftertested in liquid transformation Antibiotic cultures (μg/ml) (μg/ml)Ampicillin 5, 10, 25, 50 25 Chloramphenicol 1, 2.5, 5, 10  2.5Tetracyclin 5, 10, 20, 30 10 Erythromycin 5, 10, 20 10 Streptomycin 30Not used Kanamycin 50 Not used

Resistance of A. succinogenes to antibiotics. Growth of A. succinogenes130Z was tested in the presence of antibiotics at the concentrationslisted in Table 2. A. succinogenes 130Z grew perfectly well in thepresence of 50 μg/ml kanamycin or 30 μg/ml streptomycin. Very slowgrowth was observed in the presence of 5 μg/ml ampicillin or 5 μg/mlerythromycin (OD₆₆₀ of approximately 0.05 after 7 days culture). Nogrowth was detected in the presence of 10 μg/ml ampicillin, 1 μg/mlchloramphenicol, 5 μg/ml tetracycline, or 10 μg/ml erythromycin. A.succinogenes is sensitive to low concentrations of ampicillin,chloramphenicol, erythromycin, and tetracycline, so the correspondingresistance genes (i.e., Amp^(R), Cm^(R), Tet^(R), and ERy^(R)) could beused as selection markers on plasmids used to transform A. succinogenes.We discovered that the Tn3 Amp^(R) gene carried by pGZRS-19 is naturallyexpressed in A. succinogenes.

EXAMPLE 3

succinogenes 130Z (ATCC 55618) was used as the host strain. One hundredml of actively growing cells in TS broth (OD₆₆₀ of 0.3) were chilled onice and harvested by centrifugation at 4,000×g for 10 minutes at 4° C.Cells were washed twice with 5 ml of ice-cold 15% glycerol andresuspended in 0.2 ml of ice-cold 15% glycerol. Fifty microliters (μl)of this cell suspension was mixed with 0.5-1 microgram (μg) plasmid in achilled, 2.0 mm Gene pulser cuvette (BioRad, Richmond, Calif.).Electroporation was performed at 2.5 kV, 200 Ω, and 25 μF. Onemilliliter (ml) of TS broth was immediately added to the electroporatedcells. The suspension was incubated for 1 hour (hr) at 37° C. and thenspread (under air) on TS agar plates containing ampicillin,chloramphenicol, or tetracycline (concentrations listed in Table 2).Plates were incubated in an anaerobic jar under CO₂ atmosphere at 37° C.for 24-48 hr. A. pleuropneumoniae-E. coli shuttle vectors, pGZRS-19 andpGZRS-30 were provided by Dr. Susan West (Department of PathobiologicalSciences, School of Veterinary Medicine, University ofWisconsin-Madison, Madison, Wis. 53706). Shuttle vector UB214 wasprovided by Dr. Stefan Schwarz (für Tierzucht and Tierverhalten derBundesforschungsantalt für Landwirtschaft Braunschweig (FAL),Dörnbergstr 25-27, 29223 Celle, Germany).

Introduction of plasmids into bacteria can be done using any means knownin the art, including, but not limited to, electroporation, chemicaltransformation, conjugation, liposome mediated gene transfer, andparticle bombardment. Actinobacillaceae are naturally non-transformable.Lalonde et al. (Am. J. Vet. Res. 50:1957-1960 (1989)) were the first todevelop electroporation in A. pleuropneumoniae. Electroporation hassince been used extensively to transform various Actinobacillus,Haemophilus, and Pasteurella strains with efficiencies of up to3.10⁶-10⁷ (Brogan, J. M., et al., Gene 169:141-142(1996); Frey, J., Res.Microbiol. 143:263-269 (1992)). Conditions typically used include acapacitance set at 25 μF, a pulse controller at 200-1000 Ω, and a pulseamplitude of 2.5 to 6.5 kV/cm (Craig, F. F., et al., J. Gen. Microbiol.135:2885-2890 (1989); Dixon, L. G., et al., Plasmid 32:228-232 (1994);Frey, J., Res. Microbiol. 143:263-269 (1992); Lalonde, G., et al., Am.J. Vet. Res. 50:1957-1960 (1989); Oswald, W., et al., FEMS Microbiol.Lett. 179:153-160 (1999); West, S. E., et al., Gene 160:81-86 (1995);Wright, C. L., Plasmid 37:65-79 (1997)). The electroporation conditions(i.e., 2.5 kV, 200 Ω, and 25 μF) that are standard for E. coli, and thatare among the typical conditions used for Actinobacillaceae.

Because the organization of Actinobacillaceae promoters is differentfrom that of E. coli promoters (Doree, S. M., et al. J. Bacteriol.183:1983-1989 (2001)), most antibiotic resistance genes that have beensuccessfully used in Pasteurellaceae shuttle vectors originate fromtransposons or from Actinobacillus indigenous plasmids. We first testedampicillin, chloramphenicol, and tetracyclin resistance (Amp^(R),Cm^(R), and Tet^(R), respectively) genes that are known to be expressedin Pasteurellaceae species (Craig, F. F., et al., J. Gen. Microbiol.135:2885-2890 (1989); Kehrenberg, C., et al., Antimicrob. AgentsChemother. 42:2116-2118 (1998); West, S. E., et al., Gene 160:87-88(1995); West, W. E., et al., Gene 160:81-86 (1995); Wright, C. L., etal., Plasmid 37:65-79(1997)).

A number of plasmids have been isolated from Pasteurellaceae or havebeen constructed as vectors for Pasteurellaceae species (Craig, F. F.,et al., J. Gen. Microbiol. 135:2885-2890 (1989); Dixon, L. G., et al.,Plasmid 32:228-232 (1994); Frey, J., Res. Microbiol. 143:263-269 (1992);Galli, D. M., et al., Plasmid 36:42-48 (1996); Ishii, H., et al., NipponJuigaku Zasshi 52:1-9 (1990); Kehrenberg, C., et al., Antimicrob. AgentsChemother. 42:2116-2118 (1998); Lalonde, G., et al., Gene 85:243-246(1989); Nakano, Y., et al., Gene 169:139-140 (1996); West, S. E., etal., Gene 160:87-88 (1995); West, S. E., et al., ene 160:81-86 (1995);Wright, C. L., et al., Plasmid 37:65-79 (1997)). We tested threereplicons for their ability to replicate in A. succinogenes.

Efficiency of electroporation of A. succinogenes with plasmids pGZRS-19,pGZRS-30, and UB214 was used to determine the ability of these plasmidsto replicate and to express their antibiotic resistance genes in A.succinogenes. Electroporation of A. succinogenes with pGZRS-19 gave anaverage of 5.4×10⁴ CFU/μg plasmid (Table 4). No significant differencewas observed in the transformation yields obtained using pGZRS-19 DNApurified from E. coli and A. succinogenes strains (Table 4), suggestingthat A. succinogenes does not have a restriction system inhibitingtransformation. TABLE 4 Efficiency of electroporation of A. succinogenesand E. coli with pGZRS-19 and its derivatives CFU/μg plasmid PGZRS-19pLGZ901 pLGZ903 Host ^(a)Eco ^(b)Acs Eco Acs Eco Acs A. succinogenes130Z 6.2 × 10⁴ 5.6 × 10⁴ 5.1 × 10⁴ 7.2 × 10⁴ 2.4 × 10⁴ 4.2 × 10⁴ FZ6 5.9× 10⁴ 3.9 × 10⁴ 3.5 × 10⁴ 3.6 × 10⁴ 2.0 × 10⁴ 2.5 × 10⁴ E. coli DH5α 5.6× 10⁶ 6.3 × 10⁶ 7.2 × 10⁶ 7.3 × 10⁶ 4.7 × 10⁶ 5.4 × 10⁶ JM110 1.8 × 10⁶4.2 × 10⁶ 2.6 × 10⁶ 1.8 × 10⁶ 1.7 × 10⁶ 1.8 × 10⁶^(a)Eco: The plasmid DNA used for transformation was purified from E.coli;^(b)Acs: The plasmid DNA used for transformation was purified from A.succinogenes.

succinogenes transformants containing pGZRS-19 did not show any coloniesafter 48 hours on plates containing 100 μg/ml ampicillin, so ampicillinat 25 μg/ml was used in the selection medium. No colonies were obtainedafter transformation of A. succinogenes with pGZRS-30, using theantibiotic concentrations listed in Table 2. While the A.pleuropneumoniae-E. coli shuttle vector pGZRS-19 and its Tn3 Amp^(R)gene was successful, attempts at identifying other replicons that aremaintained and other antibiotic resistance genes that are expressed inA. succinogenes were not. The M. haemolytica pMHT1 replicon (testedusing the UB214-Amp^(R) construct) was unable to replicate in A.succinogenes. In contrast to the Tn3 Amp^(R) gene, the Tn9 Cm^(R) genecarried by pGZRS-30 and the Tet^(R) gene carried by UB214 (tested in thepGZRS-19/UB214 construct) were not expressed from their own promoters inA. succinogenes. Once expressed under a functional promoter (i.e. thepckA promoter), though, we determined that Cm^(R) and Tet^(R) werefunctional and that they could be used as selection markers in A.succinogenes. These selection markers can be used to label knock-outmutations.

Because transformation of A. succinogenes with pGZRS-19 was successful,our lack of success with pGZRS-30 most probably comes from the inabilityof the Cm^(R) gene of Tn9 to be expressed in A. succinogenes. In thecase of plasmid UB214, the absence of transformants could be due eitherto the inability of the plasmid to replicate in A. succinogenes, or tothe inability of its antibiotic resistance gene to be expressed in A.succinogenes. To determine whether UB214 (i.e., the M. haemolytica pMHT1replicon) can replicate in A succinogenes, we constructed theUB214-Amp^(R) derivative. In this plasmid, the 1.4 kb EcoRI fragmentcarrying the Amp^(R) gene of pGZRS-19 was subcloned into the uniqueEcoRI site of UB214. UB214-Amp^(R) conferred ampicillin resistance to E.coli, but A. succinogenes transformed with UB214-Amp^(R) did not showany colonies after 48 hours on ampicillin (25 μg/ml) plates. Todetermine whether the Tet^(R) gene carried by UB214 is expressed in A.succinogenes, the whole UB214 plasmid was cloned into the unique DamHIsite of pGZRS-19. The pGZRS-19/UB214 construct conferred both ampicillinand tetracycline resistance to E. coli. A. succinogenes transformed withpGZRS-19/UB214 showed many colonies after 48 hours on ampicillin (25μg/ml) plates, but none on tetracycline (5 μg/ml) plates. These resultsindicate that not only is the M. haemolytica pMHT1 replicon unable toreplicate in A. succinogenes, but its Tet^(R) gene is also not expressedfrom its own promoter in A. succinogenes.

EXAMPLE 4

Plasmid DNA purification, PCR amplification, restriction analysis,subcloning, transformation, and bacterial culture utilized methodsgenerally described in Sambrook, J., Fritsh, E. F., and Maniatis, T.,Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring HarborLaboratory Press, N.Y. (1989); and Ausubel, F. M., et al., CurrentProtocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc.,N.Y. (1993-1998). Oligonucleotides used for PCR reactions and forsequencing were synthesized by the Michigan State UniversityMacromolecular Structure, Sequencing, and Synthesis Facility. DNAsequencing was performed by the Michigan State University GenomicsTechnology Support Facility. DNA was recovered from agarose gels withthe Geneclean II kit (BIO 101, La Jolla, Calif.).

The A. succinogenes pckA gene was cloned in 4 steps. In step (i), analignment of E. coli, Anaerobiospirillum succiniciproducens, Haemophilusinfluenzae, Rhizobium sp. NGR234, and Vibrio cholerae PEPCKs sequences(Genbank accession Nos. F65135, U95960, NP_(—)438969, S18606, andQ9KNK0, respectively) indicated that sequences YGGEMKK, SEQ ID NO: 23,and NTGWNG, SEQ ID NO: 24, were highly conserved. Degenerateoligonucleotides SEQ ID NO: 1 and SEQ ID NO: 2 (Table 3) were used asprimers to amplify a pckA internal fragment using A. succinogenes 130Zchromosomal DNA as the template. The 0.7 kb PCR product was cloned intoPCR™II (Invitrogen, Carlsbad, Calif.) and sequenced. For step (ii), A.succinogenes PEPCK was purified to homogeneity as described (Shen, T.L., et al., J. Mass Spectrom. 34:1154-1165 (1999)) and submitted toN-terminal sequencing (performed by the Michigan State UniversityMacromolecular Structure, Sequencing, and Synthesis Facility).Oligonucleotides SEQ ID NO: 3 (encoding the N-terminal sequence TDLNKLV,SEQ ID NO: 25) and SEQ ID NO: 4 (in the center of the pckA gene) (Table3) were used as primers to amplify the 5′-end of pckA using A.succinogenes 130Z chromosomal DNA as the template. The 1.0 kb PCRproduct was cloned into pCR™II and sequenced. In step (iii), pckA's3′-end was amplified by an extender PCR approach (Brown, A. J. H., etal., Biotechniques 26:804-806 (1999)) as follows: A. succinogenes 130Zchromosomal DNA was digested by EcoRI, EcoRI extremities were annealedto oligonucleotides SEQ ID NO: 5 and SEQ ID NO: 6 (Table 3), 3′-endswere blocked with ddATP, and two successive amplifications wereperformed, first with primers SEQ ID NO: 7 and SEQ ID NO: 8, then withprimers SEQ ID NO: 9 and SEQ ID NO: 10 (Table 3). The final, 2.3-kb PCRproduct was cloned into pCR™II and sequenced. In step (iv), pckA'spromoter region was amplified by extender PCR. The EcoRI-digested,annealed, and blocked A. succinogenes 130Z chromosomal DNA from step(iii) was used as the template in two successive amplifications, firstwith primers SEQ ID NO: 8 and SEQ ID NO: 11, then with primers SEQ IDNO: 10 and SEQ ID NO: 12 (Table 3). The final, 2.0-kb PCR product wascloned into pCR™II and sequenced. TABLE 3 SEQ ID NO: Primer sequence (5′to 3′)^(a) Properties 1 GGTAYGGNGGNGARATGAARAARG pckA non-coding strand,encodes YGGEMKK 2 CCRTTCCANCCNGTRTT pckA non-coding strand, encodesNTGWNG 3 ATGCANGAYYTNAYAARYT pckA non-coding strand, encodes theN-terminal sequence TDLNKLV 4 TCCGCGGTTAAGAAAATCACTTT pckA codingstrand, encodes KVIFLT 5 AATTTGCATGTC Extender PCR EcoR1 linker 6TGCGAGTAAGGATCCTCACGCAAGGA Extender PCR EcoR1 adaptor ATTCCGACCAGACATGCA7 CACATCGACAACATCGTTCGTC pckA non-coding strand, encodes HIDNIVR 8TGCGAGTAAGGATCCTGACGCA Extender PCR primer P1 9 GTATTGCCGCCGGTTTCAAAACTGpckA non-coding strand, encodes LPPVSKL 10 CGCAAGGAATTCCGACCAGACAExtender PCR primer P2 11 CGACCGGTAAAAATCCCCGTATCG pckA coding strand,encodes DTGIFTGR 12 CCAAACCCGGTTTGGTTTCTTCC pckA coding strand, encodesLGLTDVKE 13 CTTTAATCGGAAGCTTTCGATAAATTG Non-coding strand, upstream ofpckA, sequence AAAATGCAG AAGCTT creates a HindIII site 14GTCAGTTCTAGATCACCTCATTGATAAT Coding strand, upstream of pckA , sequenceTCTAGA TTAAAATTAAA creates an XbaI site 15 CAATGAGGTCTAGAATGACTGACTTAAACpckA non-coding strand, sequence TCTAGA creates an AAACTCG XbaI siteupstream of the ATG start codon 16 CAAAAGCCCGGGTGGAAATACTCAGCCTT Codingstrand, downstream of pckA, sequence CCCGGG ATTTTTC creates an XmaI site17 TTTCTAGAATGCTGGCCGTCGTTTTACAAC In pUC19, sequence TCTAGA creates anXbaI site GTCGTGACTACTGG upstream of laZa's ATG start codon 18CCACATTTACCCGGGACCCCAAA AAAGAC In pUC19, sequence CCCGGG creates an XmaIsite TTAC downstream of laZa 19 AAGCTTTCTGCTAATCCTGTTACCAGTGGG InpPROEX-1, sequence AAGCTT creates a HindIII site 20AAGCTTCCGCATCAGGCGCTCTTCGCGTTC In pPROEX-1, sequence AAGCTT creates aHindIII site 31 TCTAGAATGAAATCTAACAATGCGCTC In pBR325, creates an XbaIsite upstream of Tet^(R)′s start codon 32 GAGCTCTCAGGTCGACCTGGCCCGG InpBR325, creates a SacI site downstream of Tet^(R) 33TCTAGAATGGAGAAAAAAATCACTGG In pBR325, creates an XbaI site upstream ofCm^(R)′s start codon 34 GAGCTCTTACGCCCCGCCCTGCCAC In pBR325, creates aSacI site downstream of Cm^(R)^(a)Where N is A, C, G, or T; R is A or G; and Y is T or C.

The pckA promoter region was amplified using oligonucleotides SEQ ID NO:13 and SEQ ID NO: 14 (Table 3) as primers and A. succinogeneschromosomal DNA as the template. The 230 bp PCR product was cloned intothe HindIII and XbaI sites of pGZRS19 to yield plasmid pLGZ901 (4.8 kb).The pckA gene was amplified using oligonucleotides SEQ ID NO: 15 and SEQID NO: 16 (Table 3) as primers and A. succinogenes chromosomal DNA asthe template. The 1.6 kb PCR product was cloned into the XbaI and XmaIsites of pLGZ901 to yield plasmid pLGZ902 (6.4 kb). The lacZa fragmentwas amplified using oligonucleotides SEQ ID NO: 17 and SEQ ID NO: 18(Table 3) as primers and pUC19 as the template. The 0.9 kb PCR productwas cloned into the XbaI and XmaI sites of pLGZ901 to yield plasmidpLGZ903 (5.7 kb). The ColE1 origin of replication was amplified usingoligonucleotides SEQ ID NO: 19 and SEQ ID NO: 20 (Table 3) as primersand pProEx-1 (Invitrogen, Carlsbad, Calif.) as the template. The PCRproduct (0.55 kb) was cloned into pCR™II. After sequence verification,the HindIII PCR fragment was subcloned into the HindIII site of pLGZ901,yielding plasmid pLGZ920 (5.4 kb). The Tn10 tetracycline resistance gene(Tet^(R)) was amplified using oligonucleotides SEQ ID NO: 31 and SEQ IDNO: 32 as primers and plasmid pBR325 as the template. The 1.2 kb PCRfragment was cloned into pCR2.1. After sequence verification, the PCRfragment was subcloned between the XbaI and SacI sites of pLZG920,yielding plasmid pLGZ921. The Tn9 chloramphenicol resistance gene(Cm^(R)) was amplified using oligonucleotides SEQ ID NO: 33 and SEQ IDNO: 34 as primers and plasmid pBR325 as the template. The 0.65 kb PCRfragment was cloned into pCR2.1. After sequence verification, the PCRfragment was subcloned between the XbaI and SacI sites of pLZG920,yielding plasmid pLGZ922.

The pckA gene is constitutively expressed at high levels in A.succinogenes (van der Werf, M. J., et al., Arch. Microbiol. 167:332-342(1997)), and PEPCK is a key enzyme in succinate production by A.succinogenes. For these reasons, and because we needed a strong A.succinogenes promoter that could be used in an expression vector, wedecided to clone A. succinogenes pckA. The complete sequence of A.succinogenes pckA and its promoter region is shown in FIG. 1. The 2 kbDNA fragment contained a single, 1,623 bp open reading frame, encoding a538-residue protein. In BLAST search, this protein showed 85% and 74%identity (91% and 84% similarity) to the H. influenzae and E. coli PEPCKsequences, respectively (not shown), confirming that we indeed clonedthe A. succinogenes pckA gene. As expected, the consensus sequencesinvolved in ATP, Mg²⁺, phosphoenolpyruvate, and oxaloacetate bindingthat are conserved in all ATP/ADP-dependent PEPCKs (Laivenieks, M., etal., Appl. Environ. Microbiol. 63:2273-2280 (1997)) are present in A.succinogenes PEPCK (not shown). A few nucleotides upstream of the ATGstart codon is the sequence AGGTG that could act as pckA's ribosomebinding site. Two pairs of sequences located 83 nt and 24 nt upstream ofpckA's start codon, respectively, match the A. pleuropneumoniaeconsensus promoter (Doree, S. M., et al., J. Bacteriol. 183:1983-1989(2001)) sequences TTRAA (−35) and TATAAT (−10) (FIG. 1). The distancesseparating the −35 and −10 sequences in these two potential promoterregions (i.e., 15 and 18 nucleotides) are in agreement with the shortspacing identified between the −35 and −10 elements of A.pleuropneumoniae promoters (Doree, S. M., et al., J. Bacteriol.183:1983-1989 (2001)).

EXAMPLE 5

succinogenes 130Z and E. coli DH5α cells harboring pLGZ901 and pLGZ920were inoculated in TS (A. succinogenes) and LB (E. coli) media withoutampicillin and incubated at 37° C. Culture samples were removed atone-hour intervals for numeration. Total cell number was counted onTS-agar (A. succinogenes) and LB-agar (E. coli), and plasmid-containingcells were counted on TS-agar-ampicillin (25 μg/ml, A. succinogenes) andLB-agar-ampicillin (50 μg/ml, E. coli).

The stability of pLGZ901 was tested in A. succinogenes 130Z and in E.coli DH5α. As shown in FIG. 4, pGLZ901 is much less stable in E. colithan in A. succinogenes. After five generations of growth in the absenceof antibiotic, 70% of the A. succinogenes cells still contained theplasmid, whereas fewer than 20% of the E. coli cells did. To increasethe stability of our vector in E. coli, we subcloned the ColE1 origin ofreplication into pLGZ901's unique HindIII site, yielding plasmidpLGZ920. The stability of pLGZ920 was tested in A. succinogenes 130Z andin E. coli DH5α (FIG. 4). As expected (ColE1 plasmids are typically notstable in Actinobacillaceae), the presence of the ColE1 origin ofreplication in pLGZ920 did not affect the stability of the plasmid in A.succinogenes. In contrast, pLGZ920 was significantly more stable thanpLGZ901 in E. coli: after 8 generations, while only 10% of E. coli cellsstill contained pLGZ901, 90% still contained pLGZ920.

EXAMPLE 6

β-Galactosidase activity was measured as follows: A. succinogenesrecombinant strains were grown either in TS broth or in medium A. Thirtymicroliter aliquots were harvested during the growth time course. Thecells were disrupted by vortexing after adding 0.97 ml Z-buffer (60 mMNa₂HPO₄.7H₂O, 40 mM NaH₂PO₄.H₂O, 10 mM KCl, 1 mM MgSO₄.7H₂O, and 50 mMβ-mercaptoethanol, pH 7.0), 20 μl chloroform, and 20 μl of 0.1% SDS. Theβ-galactosidase reaction was initiated by adding 0.2 ml of ONPG solution(4 mg/ml) to the cell lysate. After a thirty minute incubation at 37°C., the reaction was stopped by adding 0.5 ml of 1 M sodium carbonate.The activity was estimated from the absorbance at 420 nm.

To test PEPCK activity, A. succinogenes strains were grown in medium A,cells were harvested in the exponential phase, and they were disruptedin a French press as described (van der Werf, M. J., et al., Arch.Microbiol. 167:332-342 (1997)). The PEPCK activity was measured byfollowing the consumption of NADH in a coupled assay at 37° C. The 1 mlreaction mixture consisted of 100 mM Tris (pH 6.6), 35 mM NaHCO₃, 16 mMMgCl₂, 0.3 mM NADH, 2 U phosphoglycerate phosphokinase/glyceraldehydephosphate dehydrogenase (Sigma Diagnostics 366-2), 1 mM DTT, 10 mM ADP,1.8 mM 3-phosphoglycerate (Sigma Diagnostics 366-1), 5 mM PEP, and thecell extract. The extinction coefficient for NADH was 6.22 cm⁻¹ mM⁻¹ at340 nm. Protein concentrations were determined using the BioRad ProteinAssay kit (Richmond, Calif.) using bovine serum albumin (BSA) as thestandard.

Since we could reproducibly introduce plasmid pGZRS-19 into A.succinogenes by electroporation, and since pGZRS-19 is stably maintainedin A. succinogenes, we tested it as an expression vector in A.succinogenes. In a first step, we subcloned the pckA promoter regioninto pGZRS-19, yielding plasmid pLGZ901. Because we did not know whichof the two putative promoter regions was the pckA promoter, we subcloneda fragment that encompassed both putative promoters (FIG. 1). Thisfragment also contained the pckA putative ribosome-binding site. The A.succinogenes pckA and E. coli lacZa open reading frames were then cloneddownstream of the pckA promoter and ribosome binding site in pLGZ901,yielding plasmids pLGZ902 and pLGZ903, respectively. Electroporation ofA. succinogenes with pLGZ901 and pLGZ903 gave transformationefficiencies similar to those obtained with pGZRS-19 (Table 4).

β-Galatosidase activity was followed in cultures of A. succinogenes 130Zcomprising pLGZ903, grown in medium A and TS broth (FIG. 2).β-Galactosidase activity was up to 150 units per mg-protein after only 2hours growth. It then leveled off between 200 and 250 units permg-protein after 8 hours. No difference in activity was observed betweenthe cultures grown in glucose-based and TS media. Similarβ-galactosidase activity levels were observed in A. succinogenes FZ6comprising pLGZ903, grown in the same conditions. These results indicatethat the pckA promoter-ribosome binding site cassette allows theexpression of a foreign protein at high levels in A. succinogenes. Theseresults were verified by testing the activity of PEPCK in A.succinogenes strains 130Z and FZ6 comprising plasmid pLZG902. As seen inTable 5, 130Z and FZ6 comprising pLZG902 showed twice as much PEPCKactivity as the strains devoid of plasmid. TABLE 5 PEPCK activity in A.succinogenes strains grown in medium A PEPCK activity Strain (nmole/min· mg protein) 130Z 732 ± 26 130Z(pLZ902) 1470 ± 34  FZ6 851 ± 19FZ6(pLZ902) 1591 ± 27 

EXAMPLE 7

Expression of Tet^(R) and Cm^(R) in A. succinogenes. We need severalmarkers to conveniently engineer A. succinogenes. One can be used tolabel a gene deletion, while another one can be used to select for themaintenance of a recombinant gene on a plasmid replicon. To determinewhether, once expressed, Cm^(R) and Tet^(R) could be used as selectivemarkers in A. succinogenes, the Tn9 Cm^(R) and Tn10 Tet^(R) genes werecloned into pLGZ920 under control of the A. succinogenes pckA promoter,yielding plasmids pLGZ921 and pLGZ922, respectively. Afterelectroporation, A. succinogenes 130Z cells harboring pLGZ921 andpLGZ922 were spread on TS-agar (30 g/l TS, 10 g/l glucose) platescontaining variable amounts of tetracycline and chloramphenicol (FIG.9). Strains 130Z(pLGZ921) plated on chloramphenicol plates and130Z(pLGZ922) plated on tetracycline plates were used as the negativecontrols. As seen in FIG. 9, tetracycline-sensitive 130Z(pLGZ922) straingrew on plates containing up to 1.0 μg/ml tetracycline, but showed nogrowth after one week on plates containing higher tetracyclineconcentrations. In contrast, strain 130Z(pLGZ921) grew on platescontaining up to 5.0 μg/ml tetracycline. Chloramphenicol-sensitive130Z(pLGZ921) strain grew on plates containing up to 0.4 μg/mltetracycline, but not on plates containing higher chloramphenicolconcentrations. Strain 130Z(pLGZ922) grew on plates containing up to 5.0μg/ml chloramphenicol. These results indicate that, once under controlof the A. succinogenes pckA promoter, the Tn10 Tet^(R) and Tn9 Cm^(R)genes are expressed and functional in A. succinogenes. These resultsalso suggest that these two genes expressed under control of the pckApromoter can be used as selective markers in media containing thecorresponding antibiotic at 1.5 to 4.0 μg/ml.

While the present invention is described herein with reference toillustrated embodiments, it should be understood that the invention isnot limited hereto. Those having ordinary skill in the art and access tothe teachings herein will recognize additional modifications andembodiments within the scope thereof. Therefore, the present inventionis limited only by the Claims attached herein.

1. A plasmid comprising: (a) at least one marker gene operably linked toa first promoter functional in Actinobacillus succinogenes; (b) anorigin of replication functional in Actinobacillus succinogenes; (c) asecond promoter isolated from Actinobacillus succinogenes; and (d) acloning site downstream from the second promoter.
 2. The plasmid ofclaim 1 wherein the marker gene confers antibiotic resistance toActinobacillus succinogenes.
 3. The plasmid of claim 2 wherein theantibiotic is selected from the group consisting of ampicillin,chloramphenicol, tetracycline, and erythromycin.
 4. The plasmid of claim1 wherein the second promoter is from the pckA gene of Actinobacillussuccinogenes.
 5. The plasmid of claim 4 wherein the second promotercomprises substantially the nucleic acid sequence set forth in SEQ IDNO: 21 from between about nucleotide 25 and nucleotide
 255. 6. Theplasmid of claim 1 wherein the second promoter provides a ribosomebinding site comprising the nucleotide sequence AGGTG.
 7. The plasmid ofclaim 1 further comprising a ColE1 origin of replication.
 8. The plasmidof claim 1 wherein the cloning site comprises one or more restrictionendonuclease cleavage sites.
 9. A plasmid which is pLGZ901.
 10. Aplasmid which is pLGZ920, deposited as ATCC ______.
 11. A polypeptidewhich comprises an amino acid sequence substantially similar to theamino acid sequence set forth in SEQ ID NO.
 22. 12. A nucleic acid whichcomprises a nucleotide sequence substantially similar to the nucleotidesequence set forth in SEQ ID NO:
 21. 13. A method for producing arecombinant Actinobacillus succinogenes comprising: (a) providing aplasmid comprising at least one marker gene operably linked to a firstpromoter functional in Actinobacillus succinogenes, an origin ofreplication for the plasmid functional in Actinobacillus succinogenes, asecond promoter isolated from Actinobacillus succinogenes, and a cloningsite for a nucleic acid downstream of the second promoter; (b)transforming Actinobacillus succinogenes with the plasmid; and (c)selecting the recombinant Actinobacillus succinogenes fromnon-transformed Actinobacillus succinogenes.
 14. The method of claim 13,wherein the transformation is electroporation.
 15. The method of claim13 wherein the marker gene confers antibiotic resistance to therecombinant Actinobacillus succinogenes.
 16. The method of claim 15wherein the antibiotic is selected from the group consisting ofampicillin, tetracycline, and chloramphenicol.
 17. The method of claim15 wherein the recombinant Actinobacillus succinogenes is selected fromnon-transformed Actinobacillus succinogenes by culturing in the presenceof the antibiotic.
 18. The method of claim 13 wherein the secondpromoter is from the pckA gene of Actinobacillus succinogenes.
 19. Themethod of claim 13 wherein the second promoter comprises substantiallythe nucleic acid sequence set forth in SEQ ID NO: 21 from between aboutnucleotide 25 and nucleotide
 255. 20. The method of claim 13 wherein thesecond promoter provides a ribosome binding site comprising thenucleotide sequence AGGTG.
 21. The method of claim 13 wherein theplasmid further includes a ColE1 origin of replication.
 22. The methodof claim 13 wherein the plasmid is pLGZ901 or pLGZ920.
 23. A recombinantActinobacillus succinogenes comprising: a plasmid capable of autonomousreplication in the Actinobacillus succinogenes which comprises at leastone selectable marker gene and the Actinobacillus succinogenes pckAgene.
 24. The recombinant Actinobacillus succinogenes of claim 23wherein the pckA gene comprises the nucleic acid sequence set forth inSEQ ID NO:
 21. 25. The recombinant Actinobacillus succinogenes of claim23 wherein the plasmid is pLGZ902.
 26. The recombinant Actinobacillussuccinogenes of claim 23 wherein the marker gene confers an antibioticresistance selected from the group consisting of ampicillin,tetracycline, and chloramphenicol to the recombinant Actinobacillussuccinogenes.
 27. A method for producing succinate comprising: (a)providing a recombinant Actinobacillus succinogenes comprising a plasmidcapable of autonomous replication in Actinobacillus succinogenes whichcomprises at least one selectable marker gene and a recombinant geneexpressed under the control of the Actinobacillus succinogenes pckApromoter; (b) providing a growth medium for culturing the recombinantActinobacillus succinogenes; and (c) culturing the recombinantActinobacillus succinogenes in the growth medium to produce thesuccinate.
 28. The method of claim 27 wherein the promoter comprisessubstantially the nucleic acid sequence set forth in SEQ ID NO: 21 frombetween about nucleotide 25 and nucleotide
 255. 29. The method of claim27 wherein the plasmid is selected from the group consisting of pLGZ901and pLGZ920.
 30. The method of claim 27 wherein the marker gene confersresistance to an antibiotic selected from the group of ampicillin,tetracycline, and chloramphenicol to the recombinant Actinobacillussuccinogenes.
 31. The method of claim 28 wherein the growth mediumcomprises an antibiotic selected from the group of ampicillin,tetracycline, and chloramphenicol.
 32. The method of claim 27 forproducing succinate wherein the recombinant Actinobacillus succinogenesis a mutant strain in which an endogenous gene which inhibits succinateproduction comprises a deletion.
 33. The method of claim 32 whereinendogenous gene further comprises a selectable marker under the controlof the Actinobacillus succinogenes pckA promoter.
 34. The method ofclaim 33 wherein the selectable marker gene confers resistance to anantibiotic selected from the group consisting of chloramphenicol andtetracycline to the mutant Actinobacillus succinogenes strain.
 35. Arecombinant microorganism comprising the nucleic acid which comprises anucleotide sequence substantially similar to the nucleotide sequence setforth in SEQ ID NO:
 21. 36. A plasmid which is pLGZ921.
 37. A plasmidwhich is pLGZ922.